(Invited) COVID-19 As a Technology Accelerator for High-Speed Polymerase Chain Reaction (PCR)

Abstract

Limit of detection (LOD), speed and cost for some of the most important diagnostics tools for COVID-19 i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) all benefitted from both the financial and regulatory support brought about by the pandemic. Before COVID-19, LFA was the fastest and least expensive but came with the poorest limit of detection (LOD). The slowest, most expensive but with the best (lowest) LOD was PCR. In between those two extremes, in terms of cost, LOD and speed, was an ELISA . Today, through the “COVID-19 accelerator effect” there are point of care (POC) reverse transcriptase quantitative PCR (ie., RT-qPCR) COVID-19 tests on the market that provide sample to answer in under 20 minutes, while retaining the same very low LOD but at a lower cost [1]. There are even POC PCR tests under development that come in at less than 10 minutes: beating both LFA and ELISAs in terms of time and LOD [2]. Although RT-qPCR has gained the most in overall performance, signal-amplified POC LFAs are possible now with an LOD that rivals that of an ELISA.However, implementing the next generation PCR equipment in point-of-care settings, remains very challenging because of difficulties in reaching a 10 minute or below sample to answer time , instrument complexity and cost. In this work, we discuss the engineering challenges involved in the development of high-speed microfluidic PCR equipment. To guide this review/comparison of PCR advances we introduce three parameters. The first one is the overall sample to answer time (t), the second is called lambda (λ) and is a measure of the degree to which stochastic effects interfere with obtaining analytical results and thereby setting the minimal number of copies required per volume. The third parameter gamma (γ), introduced first by Dong et al [3], is the ratio of thermal cycling time (in min) to the heat cycled sample volume (in mL). It describes the trade-off between the speed of the PCR system and the desired level of sensitivity. It can be interpreted as a system efficiency: the most efficient system heat cycles the largest amount of sample in the smallest amount of time.With these three criteria in mind we set out to suggest designs for an optimized POC reverse transcriptase (RT) quantitative qPCR or RT-qPCR system. Applications of RT-qPCR include quantifying gene expression levels, validating RNA interference (RNAi), and detecting pathogens such as viruses. The aim of this specific POC RT-qPCR system is to provide sample to answer in ten minutes or less for a respiratory pathogens panel, with an LOD of 500 copies/mL and the capability to multiplex (8 chambers) while remaining portable and low cost.Finally we introduce two of the most daunting, remaining, microfluidics challenges that both need to be tackled to implement an ideal microfluidic platform like the POC RT-qPCR system described above. One is liquid storage and the other is proportional valves on a disposable microfluidic platform. Liquid storage is essential when the assay requires repeated washes, if these washes can come from one and the same reservoir via a proportional valve, real estate is gained and system complexity is reduced. The storage issue is further complicated when the design also calls for storing lyophilized or dried reagents on the same disposable. We compare some of the most recent approaches to liquid storage and proportional valving that we believe will enable the fast POC diagnostic platforms of the future. 1. Tsang, Hin Fung, Wai Ming Stanley Leung, Lawrence Wing Chi Chan, William Chi Shing Cho, and Sze Chuen Cesar Wong. “Performance Comparison of the Cobas® Liat® and Cepheid® GeneXpert® Systems on SARS-CoV-2 Detection in Nasopharyngeal Swab and Posterior Oropharyngeal Saliva.” Expert Review of Molecular Diagnostics 21, no. 5 (May 4, 2021): 515–18. 2. BUCKLAND, Justin, Tom JELLICOE, Alex STOKOE, and Amaru ARAYA-WILLIAMS. “VARIABLE TEMPERATURE REACTOR, HEATER AND CONTROL CIRCUIT FOR THE SAME.” Patent Application, January 30, 2020. 3. Dong, Xiaobin, Luyao Liu, Yunping Tu, Jing Zhang, Guijun Miao, Lulu Zhang, Shengxiang Ge, Ningshao Xia, Duli Yu, and Xianbo Qiu. “Rapid PCR Powered by Microfluidics: A Quick Review under the Background of COVID-19 Pandemic.” Trends in Analytical Chemistry 143 (October 2021): 116377.